Peptide epitopes of apolipoprotein b

ABSTRACT

The present invention relates to antibodies raised against fragments of apolipoprotein B, in particular defined peptides thereof, for immunization or therapeutic treatment of mammals, including humans, against ischemic cardiovascular diseases, using one or more of said antibodies.

PRIORITY INFORMATION

This application is a divisional of U.S. Utility application Ser. No.10/115,072, filed Apr. 4, 2002, which claims priority to SwedishApplication Nos. 0101232-7, filed on Apr. 5, 2001, and 0103754-8, filedNov. 9, 2001, and the benefit of U.S. Provisional Application Ser. No.60/281,410, filed Apr. 5, 2001, the content of which is incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new peptides, in particular peptides tobe used for immunization therapy for treatment of atherosclerosis, andfor development of peptide based ELISA for the determination of immuneresponse against oxidized low density lipoprotein and the diagnosis ofthe presence or absence of atherosclerosis.

2. Brief Description of the Art

In particular the invention includes:

-   1) The use of any of the peptides listed in table 1, alone or in    combination, native or MDA-modified, preferably together with a    suitable carrier and adjuvant as an immunotherapy or    “anti-atherosclerosis “vaccine” for prevention and treatment of    ischemic-   2) cardiovascular disease.-   3) The use of the same peptides in ELISA for detection of antibodies    related to increased or decreased risk of development of ischemic    cardiovascular diseases.

Atherosclerosis is a chronic disease that causes a thickening of theinnermost layer (the intima) of large and medium-sized arteries. Itdecreases blood flow and may cause ischemia and tissue destruction inorgans supplied by the affected vessel. Atherosclerosis is the majorcause of cardiovascular disease including myocardial infarction, strokeand peripheral artery disease. It is the major cause of death in thewestern world and is predicted to become the leading cause of death inthe entire world within two decades.

The disease is initiated by accumulation of lipoproteins, primarilylow-density lipoprotein (LDL), in the extracellular matrix of thevessel. These LDL particles aggregate and undergo oxidativemodification. Oxidized LDL is toxic and cause vascular injury.Atherosclerosis represents in many respects a response to this injuryincluding inflammation and fibrosis.

In 1989 Palinski and coworkers identified circulating autoantibodiesagainst oxidized LDL in humans. This observation suggested thatatherosclerosis may be an autoimmune disease caused by immune reactionsagainst oxidized lipoproteins. At this time several laboratories begansearching for associations between antibody titers against oxidized LDLand cardiovascular disease. However, the picture that emerged from thesestudies was far from clear. Antibodies existed against a large number ofdifferent epitopes in oxidized LDL, but the structure of these epitopeswas unknown. The term “oxidized LDL antibodies” thus referred to anunknown mixture of different antibodies rather than to one specificantibody. T cell-independent IgM antibodies were more frequent thanT-cell dependent IgG antibodies.

Antibodies against oxidized LDL were present in both patients withcardiovascular disease and in healthy controls. Although some earlystudies reported associations between oxidized LDL antibody titers andcardiovascular disease, others were unable to find such associations. Amajor weakness of these studies was that the ELISA tests used todetermine antibody titers used oxidized LDL particles as ligand. LDLcomposition is different in different individuals, the degree ofoxidative modification is difficult both to control and assess andlevels of antibodies against the different epitopes in the oxidized LDLparticles can not be determined. To some extent, due to the technicalproblems it has been difficult to evaluate the role of antibodyresponses against oxidized LDL using the techniques available so far,but, however, it is not possible to create well defined and reproduciblecomponents of a vaccine if one should use intact oxidized LDL particles.

Another way to investigate the possibility that autoimmune reactionsagainst oxidized LDL in the vascular wall play a key role in thedevelopment of atherosclerosis is to immunize animals against its ownoxidized LDL. The idea behind this approach is that if autoimmunereactions against oxidized LDL are reinforced using classicalimmunization techniques this would result in increased vascularinflammation and progressive of atherosclerosis. To test this hypothesisrabbits were immunized with homologous oxidized LDL and then inducedatherosclerosis by feeding the animals a high-cholesterol diet for 3months.

However, in contrast to the original hypothesis immunization withoxidized LDL had a protective effect reducing atherosclerosis with about50%. Similar results were also obtained in a subsequent study in whichthe high-cholesterol diet was combined with vascular balloon-injury toproduce a more aggressive plaque development. In parallel with ourstudies several other laboratories reported similar observations. Takentogether the available data clearly demonstrates that there exist immunereactions that protect against the development of atherosclerosis andthat these involves autoimmunity against oxidized LDL.

These observations also suggest the possibility of developing an immunetherapy or “vaccine” for treatment of atherosclerosis-basedcardiovascular disease in man. One approach to do this would be toimmunize an individual with his own LDL after it has been oxidized byexposure to for example copper. However, this approach is complicated bythe fact that it is not known which structure in oxidized LDL that isresponsible for inducing the protective immunity and if oxidized LDLalso may contain epitopes that may give rise to adverse immunereactions.

The identification of epitopes in oxidized LDL is important for severalaspects:

First, one or several of these epitopes are likely to be responsible foractivating the anti-atherogenic immune response observed in animalsimmunized with oxidized LDL. Peptides containing these epitopes maytherefore represent a possibility for development of an immune therapyor “atherosclerosis vaccine” in man. Further, they can be used fortherapeutic treatment of atheroschlerosis developed in man.Secondly, peptides containing the identified epitopes can be used todevelop ELISAs able to detect antibodies against specific structure inoxidized LDL. Such ELISAs would be more precise and reliable than onespresently available using oxidized LDL particles as antigen. It wouldalso allow the analyses of immune responses against different epitopesin oxidized LDL associated with cardiovascular disease.

U.S. Pat. No. 5,972,890 relates to a use of peptides for diagnosingatherosclerosis. The technique presented in said US patent is as aprinciple a form of radiophysical diagnosis. A peptide sequence isradioactively labelled and is injected into the bloodstream. If thispeptide sequence should be identical with sequences present inapolipoprotein B it will bind to the tissue where there are receptorspresent for apolipoprotein B. In vessels this is above allatherosclerotic plaque. The concentration of radioactivity in the wallof the vessel can then be determined e.g., by means of a gamma camera.The technique is thus a radiophysical diagnostic method based on thatradioactively labelled peptide sequences will bound to their normaltissue receptors present in atherosclerotic plaque and are detectedusing an external radioactivity analysis. It is a direct analysis methodto identify atherosclerotic plaque. It requires that the patient begiven radioactive compounds.

SUMMARY OF THE INVENTION

The technique of the present invention is based on quite differentprinciples and methods. In accordance with claim 1 the invention relatesto fragments of apolipoprotein B for immunisation against cardiovasculardisease as well as a method for diagnosing immuno reactions againstpeptide sequences of apolipoprotein B. Such immuno reactions have inturn showed to be increased in individuals having a developedatherosclerosis. The present technique is based in attaching peptidesequences in the bottom of polymer wells. When a blood sample is addedthe peptides will bind antibodies, which are specific to thesesequences. The amount of antibodies bound is then determined using animmunological method/technique. In contrast to the technique of said USpatent this is thus not a direct determination method to identify andlocalise atherosclerotic plaque but determines an immunologicalresponse, which shows a high degree of co-variation with the extensionof the atherosclerosis.

The basic principle of the present invention is thus quite differentfrom that of said patent. The latter depends on binding of peptidesequences to the normal receptors of the lipoproteins present inatherosclerotic tissue, while the former is based on the discovery ofimmuno reactions against peptide sequences and determination ofantibodies to these peptide sequences.

Published studies (Palinski et al., 1995, and George et al., 1998) haveshown that immunisation against oxidised LDL reduces the development ofatherosclerosis. This would indicate that immuno reactions againstoxidised LDL in general have a protecting effect. The results givenherein have, however, surprisingly shown that this is not always thecase. E.g., immunisation using a mixture of peptides #10, 45, 154, 199,and 240 gave rise to an increase of the development of atherosclerosis.Immunisation using other peptide sequences, e.g., peptide sequences #1,and 30 to 34 lacks total effect on the development of atherosclerosis.The results are surprising because they provide basis for the fact thatimmuno reactions against oxidised LDL, can protect against thedevelopment, contribute to the development of atherosclerosis, and bewithout any effect at all depending on which structures in oxidised LDLthey are directed to. These findings make it possible to developimmunisation methods, which isolate the activation of protecting immunoreactions. Further, they show that immunisation using intact oxidisedLDL could have a detrimental effect if the particles used contain a highlevel of structures that give rise to atherogenic immuno reactions.

WO 99/08109 relates to the use of a panel of monoclonal mouseantibodies, which bind to particles of oxidised LDL in order todetermine the presence of oxidised LDL in serum and plasma. This is thustotally different from the present invention wherein a method fordetermining antibodies against oxidised LDL is disclosed.

U.S. Pat. No. 4,970,144 relates to a method for preparing antibodies bymeans of immunisation using peptide sequences, which antibodies can beused for the determination of apolipoproteins using ELISA. This is thussomething further quite different from the present invention.

U.S. Pat. No. 5,861,276 describes a recombinant antibody to the normalform of apolipoprotein B. This antibody is used for determining thepresence of normal apolipoprotein B in plasma and serum, and fortreating atherosclerosis by lowering the amount of particles of normalLDL in the circulation.

Thus in the present invention the use of antibodies are described fortreating atherosclerosis. However, contrary to the U.S. Pat. No.5,861,276, these antibodies are directed to structures present inparticles of oxidised LDL and not to the normal particle of LDL. Theadvantage is that it is the oxidised LDL, which is supposed to give riseto the development of atherosclerosis. The use of antibodies directed tostructures being specific to oxidised LDL is not described in said USpatent.

Oxidation of lipoproteins, mainly LDL, in the arterial wall is believedto be an important factor in the development of atherosclerosis.Products generated during oxidation of LDL are toxic to vascular cells,cause inflammation and initiate plaque formation. Epitopes in oxidizedLDL are recognized by the immune system and give rise to antibodyformation. Animal experiments have shown that some of these immuneresponses have a protective effect against atherosclerosis. Antibodiesare generally almost exclusively directed against peptide-basedstructures. Using a polypeptide library covering the complete sequenceof the only protein present in LDL, apolipoprotein B, the epitopes havebeen identified in oxidized LDL that give rise to antibody formation inman. These peptide-epitopes can be used to develop ELISAs to studyassociations between immune responses against oxidized LDL andcardiovascular disease and to develop an immunotherapy oranti-atherosclerosis “vaccine” for prevention and treatment of ischemiccardiovascular disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-6 show antibody response to the different peptides prepared inaccordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

A molecular characterization of the epitopes in oxidized LDL has beenperformed that give rise to antibody-dependent immune responses in man.The approach used takes advantage of the fact that immune reactionsalmost exclusively are directed against 5-6 amino acid long peptidesequences. LDL only contains one protein, the 4563 amino acid longapolipoprotein B. During oxidation apolipoprotein B is fragmented andaldehyde adducts coupled to positively charged amino acids, inparticularly lysine. This means that peptide sequences not normallyexposed because of the three dimensional structure of apolipoprotein Bbecome accessible to immune cells and/or that normally exposed peptidesequences becomes immunogenic because haptenization with aldehydes.

It has thereby been determined that the following peptides, native orMDA derivatives possess such an efficiency as producing animmuno-response. These peptides are:

FLDTVYGNCSTHFTVKTRKG, (SEQ ID NO: 1) PQCSTHILQWLKRVHANPLL, (SEQ ID NO:2) VISIPRLQAEARSEILAHWS, (SEQ ID NO: 3) KLVKEALKESQLPTVMDFRK, (SEQ IDNO: 4) LKFVTQAEGAKQTEATMTFK, (SEQ ID NO: 5) DGSLRHKFLDSNIKFSHVEK, (SEQID NO: 6) KGTYGLSCQRDPNTGRLNGE, (SEQ ID NO: 7) RLNGESNLRFNSSYLQGTNQ,(SEQ ID NO: 8) SLTSTSDLQSGIIKNTASLK, (SEQ ID NO: 9)TASLKYENYELTLKSDTNGK, (SEQ ID NO: 10) DMTFSKQNALLRSEYQADYE, (SEQ ID NO:11) MKVKIIRTIDQMQNSELQWP, (SEQ ID NO: 12) IALDDAKINFNEKLSQLQTY, (SEQ IDNO: 13) KTTKQSFDLSVKAQYKKNKH, (SEQ ID NO: 14) EEEMLENVSLVCPKDATRFK, (SEQID NO: 15) GSTSHHLVSRKSISAALEHK, (SEQ ID NO: 16) IENIDFNKSGSSTASWIQNV,(SEQ ID NO: 17) IREVTQRLNGEIQALELPQK, (SEQ ID NO: 18)EVDVLTKYSQPEDSLIPFFE, (SEQ ID NO: 19) HTFLIYITELLKKLQSTTVM, (SEQ ID NO:20) LLDIANYLMEQIQDDCTGDE, (SEQ ID NO: 21) CTGDEDYTYKIKRVIGNMGQ, (SEQ IDNO: 22) GNMGQTMEQLTPELKSSILK, (SEQ ID NO: 23) SSILKCVQSTKPSLMIQKAA, (SEQID NO: 24) IQKAAIQALRKMEPKDKDQE, (SEQ ID NO: 25) RLNGESNLRFNSSYLQGTNO,(SEQ ID NO: 26) SLNSHGLELNADILGTDKIN, (SEQ ID NO: 27)WIQNVDTKYQIRIQIQEKLQ, (SEQ ID NO: 28) TYISDWWTLAAKNLTDFAEQ, (SEQ ID NO:29) EATLQRIYSLWEHSTKNHLQ, (SEQ ID NO: 30) ALLVPPETEEAKQVLFLDTV, (SEQ IDNO: 31) IEIGLEGKGFEPTLEALFGK, (SEQ ID NO: 32) SGASMKLTTNGRFREHNAKF, (SEQID NO: 33) NLIGDFEVAEKINAFRAKVH, (SEQ ID NO: 34) GHSVLTAKGMALFGEGKAEF,(SEQ ID NO: 35) FKSSVITLNTNAELFNQSDI, (SEQ ID NO: 36)FPDLGQEVALNANTKNQKIR, (SEQ ID NO: 37)as well as the non antibody-producing peptide ATRFKHLRKYTYNYQAQSSS (SEQID NO: 38),or an active site of one or more of these peptides.

Material and Methods

To determine which parts of apolipoprotein B that become immunogenic asa result of LDL oxidation a polypeptide library consisting of 20 aminoacid long peptides covering the complete human apolipoprotein B sequencewas produced. The peptides were produced with a 5 amino acid overlap tocover all sequences at break points. Peptides were used in their nativestate, or after incorporation in phospholipid liposomes, afteroxidization by exposure to copper or after malone dealdehyde(MDA)-modification to mimic the different modifications of the aminoacids that may occur during oxidation of LDL.

Peptides

The 302 peptides corresponding to the entire human apolipoprotein Bamino acid sequence were synthesized (Euro-Diagnostica AB, Malmö, Swedenand K J Ross Petersen A S, Horsholm, Denmark) and used in ELISA. Afraction of each synthetic peptide was modified by 0.5 M MDA(Sigma-Aldrich Sweden AB, Stockholm, Sweden) for 3 h at 37° C. and inpresence of liposomes by 0.5 M MDA for 3 h at 37° C. or by 5 μM CuCl₂(Sigma) for 18 h at 37° C. The MDA-modified peptides were dialyzedagainst PBS containing 1 mM EDTA with several changes for 18 h at 4° C.The modification of the peptides was tested in denatured polyacrylamidegels (Bio-Rad Laboratories, Hercules, Calif.), suitable for separationof peptides. Peptides were numbered 1-302 starting at the N-terminal endof the protein.

Other aldehydes can be used for preparing derivatives, suchhydroxynonenal and others.

Liposomes

A mixture of egg phosphatidylcholine (EPC) (Sigma) andphosphatidylserine (PS) (Sigma) in a chloroform solution at a molarratio of 9:1 and a concentration of 3 mM phospholipid (PL) wasevaporated in a glass container under gentle argon stream. The containerwas then placed under vacuum for 3 hours. A solution containing 0.10 mMpeptide (5 ml) in sterile filtered 10 mM HEPES buffer pH 7.4, 145 mMNaCl and 0.003% sodium azide was added to the EPC/PS dried film andincubated for 15 min at 50° C. The mixture was gently vortex for about 5min at room temperature and then placed in ice-cold water bath andsonicated with 7.5 amplitude microns for 3×3 min (Sonyprep 150 MSESanyo, Tamro-Medlab, Sweden) with 1 min interruptions. The PL-peptidemixture, native or modified by 0.5 M MDA for 3 h at 37° C. or 5 mM CuCl₂for 18 h at 37° C., was stored under argon in glass vials at 4° C.wrapped in aluminum foil and used within 1 week. The MDA-modifiedmixture was dialyzed against PBS containing 1 mM EDTA with severalchanges for 18 h at 4° C. before storage. The modification of themixture was tested in denatured polyacrylamide gels (Bio-RadLaboratories AB, Sundbyberg, Sweden), suitable for separation ofpeptides.

Plasma Samples

Plasma samples from 10 patients with cardiovascular disease (AHP) and 50plasma samples, 25 women and 25 men, from normal blood donors (NHP) werecollected and pooled. The two pools were aliquoted and stored in −80° C.

ELISA

Native or modified synthetic peptides diluted in PBS pH 7.4 (20 μg/ml),in presence or absence of liposomes, were absorbed to microtiter platewells (Nunc MaxiSorp, Nunc, Roskilde, Denmark) in an overnightincubation at 4° C. As a reference, one of the peptides (P6) was run oneach plate. After washing with PBS containing 0.05% Tween-20 (PBS-T) thecoated plates were blocked with SuperBlock in TBS (Pierce, Rockford,Ill.) for 5 min at room temperature followed by an incubation of pooledhuman plasma, AHP or NHP, diluted 1/100 in TBS-0.05% Tween-20 (TBS-T)for 2 h at room temperature and then overnight at 4° C. After rinsing,deposition of auto-antibodies directed to the peptides were detected byusing biotinylated rabbit anti-human IgG- or IgM-antibodies (Dako A/S,Glostrup, Denmark) appropriately diluted in TBS-T. After anotherincubation for 2 h at room temperature the plates were washed and thebound biotinylated antibodies were detected by alkaline phosphataseconjugated streptavidin (Sigma), incubated for 2 h at room temperature.The color reaction was developed by using phosphatase substrate kit(Pierce) and the absorbance at 405 nm was measured after 1 h ofincubation at room temperature. The absorbance values of the differentpeptides were divided with the absorbance value of P6 and compared.

The sequences in apolipoprotein B that were recognized by antibodies inhuman plasma are shown as Seq. Id 1-37 in the accompanying drawing. BothAHP and NHP contained antibodies to a large number of differentpeptides. Antibodies against both native and modified peptides wereidentified. Generally antibody titers to MDA modified peptides werehigher or equal to that of the corresponding native peptide. Comparisonbetween native, MDA-modified, copper-oxidized peptide showed a highdegree of correlation and that the highest antibody titers were detectedusing MDA-modified peptides. The use of peptides incorporated intoliposomes did not result in increased antibody levels. Antibodies of theIgM subclass were more common than antibodies of the IgG subtype.

The peptides against which the highest antibody levels were detectedcould be divided into six groups with common characteristics (Table 1):

(A) High levels of IgG antibodies to MDA-modified peptides (n=3).(B) High levels of IgM antibodies, but no difference between native andMDA-modified peptides (n=9).(C) High levels of IgG antibodies, but no difference between native andMDA-modified peptides (n=2).(D) High levels of IgG antibodies to MDA-modified peptides and at leasttwice as much antibodies in the NHP-pool as compared to the AHP-pool(n=5).(E) High levels of IgM antibodies to MDA-modified peptides and at leasttwice as much antibodies in the NHP-pool as compared to the AHP-pool(n=11)(F) High levels of IgG antibodies, but no difference between intact andMDA-modified peptides but at least twice as much antibodies in theAHP-pool as compared to the NHP-pool (n=7).(G) No level of IgG or IgM antibodies

TABLE 1 A. High IgG, MDA-difference P 11. FLDTVYGNCSTHFTVKTRKG, (SEQ IDNO: 1) P 25. PQCSTHILQWLKRVHANPLL, (SEQ ID NO: 2) P 74.VISIPRLQAEARSEILAHWS, (SEQ ID NO: 3) B. High IgM, no MDA-difference P40. KLVKEALKESQLPTVMDFRK, (SEQ ID NO: 4) P 68. LKFVTQAEGAKQTEATMTFK,(SEQ ID NO: 5) P 94. DGSLRHKFLDSNIKFSHVEK, (SEQ ID NO: 6) P 99.KGTYGLSCQRDPNTGRLNGE, (SEQ ID NO: 7) P 100. RLNGESNLRFNSSYLQGTNQ, (SEQID NO: 8) P 102. SLTSTSDLQSGIIKNTASLK, (SEQ ID NO: 9) P 103.TASLKYENYELTLKSDTNGK, (SEQ ID NO: 10) P 105. DMTFSKQNALLRSEYQADYE, (SEQID NO: 11) P 177. MKVKIIRTIDQMQNSELQWP, (SEQ ID NO: 12) C. High IgG, noMDA difference P 143. IALDDAKINFNEKLSQLQTY, (SEQ ID NO: 13) P 210.KTTKQSFDLSVKAQYKKNKH, (SEQ ID NO: 14) D. NHS/AHP, IgG-ak > 2,MDA-difference P1. EEEMLENVSLVCPKDATRFK, (SEQ ID NO: 15) P 129.GSTSHHLVSRKSISAALEHK, (SEQ ID NO: 16) P 148. IENIDFNKSGSSTASWIQNV, (SEQID NO: 17) P 162. IREVTQRLNGEIQALELPQK, (SEQ ID NO: 18) P 252.EVDVLTKYSQPEDSLIPFFE, (SEQ ID NO: 19) E. NHS/AHP, IgM-ak > 2,MDA-difference P 301. HTFLIYITELLKKLQSTTVM, (SEQ ID NO: 20) P 30.LLDIANYLMEQIQDDCTGDE, (SEQ ID NO: 21) P 31. CTGDEDYTYKIKRVIGNMGQ, (SEQID NO: 22) P 32. GNMGQTMEQLTPELKSSILK, (SEQ ID NO: 23) P 33.SSILKCVQSTKPSLMIQKAA, (SEQ ID NO: 24) P 34. IQKAAIQALRKMEPKDKDQE, (SEQID NO: 25) p 100. RLNGESNLRFNSSYLQGTNQ, (SEQ ID NO: 26) P 107.SLNSHGLELNADILGTDKIN, (SEQ ID NO: 27) P 149. WIQNVDTKYQIRIQIQEKLQ, (SEQID NO: 28) P 169. TYISDWWTLAAKNLTDFAEQ, (SEQ ID NO: 29) P 236.EATLQRIYSLWEHSTKNHLQ, (SEQ ID NO: 30) F. NHS/AHP, IgG-ak < 0.5,noMDA-difference P 10. ALLVPPETEEAKQVLFLDTV, (SEQ ID NO: 31) P 45.IEIGLEGKGFEPTLEALFGK, (SEQ ID NO: 32) P 111. SGASMKLTTNGRFREHNAKF, (SEQID NO: 33) P 154. NLIGDFEVAEKINAFRAKVH, (SEQ ID NO: 34) P 199.GHSVLTAKGMALFGEGKAEF, (SEQ ID NO: 35) P 222. FKSSVITLNTNAELFNQSDI, (SEQID NO: 36) P 240. FPDLGQEVALNANTKNQKIR, (SEQ ID NO: 37) G. P 2.ATRFKHLRKYTYNYQAQSSS. (SEQ ID NO: 38)

All of these 38 peptide sequences represent targets for immune reactionsthat may be of importance for the development of atherosclerosis andischemic cardiovascular diseases. These peptides may therefor be used todevelop ELISAs to determine the associations between antibody levelsagainst defined sequences of MDA-modified amino acids in apolipoproteinB and risk for development of cardiovascular disease.

These peptides also represent possible mediators of the protectiveimmunity observed in experimental animals immunized with oxidized LDLand may be used for testing in further development of an immunizationtherapy or “vaccine” against atherosclerosis.

Thus 38 different sequences in the human apolipoprotein B protein havebeen identified that give rise to significant immune responses in man.These epitopes are likely to represent what has previously beendescribed as antibodies to oxidized LDL. Since most immune responses aredirected against peptide sequences and apolipoprotein B is the onlyprotein in LDL the approach used in this project should be able toidentify the specific epitopes for essentially all antibodies againstoxidized LDL-particles. A family of phospholipid specific antibodiesincluding antibodies against cardiolipin has been described to reactwith oxidized LDL but the specificity and role of these antibodiesremain to be fully characterized.

In many cases antibody titers were higher to MDA-modified polypeptidesthan to native sequences. If antibodies were detected against a MDAmodified sequence it was almost always associated with presence ofantibodies against the native sequence. A likely explanation to this isthat the immune response against an MDA-modified amino acid sequence inapolipoprotein B (the MDA-modification occurring as a result of LDLoxidation) leads to a break of tolerance against the native sequence.For other sequences there was no difference in antibody titers againstMDA-modified or native sequences. This would suggest that the immunereactions are directed against the native sequences. There should be noimmune response against amino acid sequences in protein normally exposedto the immune system. In the native LDL particle large parts of theapolipoprotein B protein is hidden in phospholipid layer of LDL andtherefore not accessible for the immune system. During oxidation of LDLthe apolipoprotein B amino acid chain is fragmented leading to changesin the three-dimensional structure. This is likely to lead to exposureof peptide sequences normally not accessible for the immune system andto generation of antibodies against these sequences which may explainthe presence of antibodies against native apolipoprotein B sequencesobserved. Alternatively, the true immune response is againstMDA-modified sequences but the cross-reactivity with native sequences isso great that no difference in binding can be demonstrated.

TABLE 2 Associations between antibodies to different peptides andatherosclerosis in the carotid artery assessed as intima/media thicknessin 78 subjects (26 subjects who later developed myocardial infarction,26 healthy controls and 26 high-risk individuals without disease). IgGIgM Peptide Native MDA-modified Native MDA-modified 301 + 10 + + 11 ++ +25 + + ++ +++ 30 ++ 31 ++

32

33 + 34 + 45 ++ ++ +++ 74 ++ + + ++ 99 100 + ++ 102

103 + 105 129 ++ +++ 143 + + ++ + 148 + 154 +++ ++ 162 + ++ 199 210 +240 ++

+, r > 0.2 < 0.3, p = <0.05; ++, r > 0.3 < 0.4, p = 0.01; +++, r > 0.4,p = <0.001, grey, peptide antibody levels significantly increased in thegroup suffering from myocardial infarction.

The possibility that the ELISAs based on these peptides (native orMDA-modified) can be used to determine associations between immunereaction against defined epitopes in oxidized LDL and presence and/orrisk for development of cardio-vascular disease was investigated in apilot study. The study was performed on subjects participating in theMalmö Diet Cancer study a population based study in which over 30,000individuals were recruited between 1989 and 1993. Antibody levelsagainst the 24 out of 38 peptides listed in Table 1 were determined inbase line plasma samples of 26 subjects who developed an acutemyocardial infarction during the follow-up period and 26 healthycontrols matched for age, gender and smoking. An additional group of 26subjects, matched for age, gender, and smoking, but all with LDLcholesterol levels above 5.0 mmol/L was also included to study antibodylevels in a high-risk group that has not developed cardiovasculardisease.

For 19 out of the 24 peptides analyzed, significant correlations wereidentified between IgM antibody levels against MDA-modified peptides andthe severity of atherosclerosis in the carotid artery (intima/mediathickness) as assessed by ultrasound investigation of common carotidartery, i.e., the higher antibody levels the more atherosclerosis (Table2). For many of these peptides significant correlations also existedbetween antibody levels to native peptides and carotid intima/mediathickness. Only 4 peptides showed a significant correlation between IgGantibodies and carotid intima/media thickness. These observationssuggest that ELISA using these MDA-modified peptides (alone or incombination) may be used to identify subjects with increasedatherosclerosis.

Four of the peptides tested were not only associated with increasedpresence of atherosclerosis but were also significant elevated in thegroup of subjects that later suffered from a myocardial infarction(Table 2). Data for one of these peptides (peptide 240) is shown in FIG.7. These observations also demonstrate that peptide-based ELISA also maybe used to identify subjects with an increased risk to developmyocardial infarction.

There were also significant increases in IgG antibody levels for nativepeptides 103, 162 and 199, as well as MDA modified 102 in the group thatlater suffered from myocardial infarction. However, the IgG antibodiesagainst these peptides were not significantly associated with thepresence of atherosclerosis in the carotid artery.

A particularly interesting observation was made with antibodies againstMDA-modified peptide 210 for which there was significantly higher levelsof IgM antibodies in the healthy controls and the high-risk group (LDLcholesterol above 5.0 mmol/L) than in the group that developed amyocardial infarction. Accordingly antibodies against MDA-modifiedpeptide 210 may represent a marker for individuals with a decreased riskto develop cardiovascular disease.

It has now been demonstrated that immunization with native andMDA-modified apo B-100 peptide sequences results in an inhibition ofatherosclerosis in experimental animals (Nordin Fredrikson, Söderberg etal, Chyu et al). The mechanisms through which these athero-protectiveimmune responses operate remain to be fully elucidated. However, onelikely possibility is that the athero-protective effect is mediated byantibodies generated against these peptides sequences. These antibodiescould, for example facilitate the removal of oxidatively damaged LDLparticles by macrophage Fc receptors.

Macrophage scavenger receptors only recognize LDL with extensiveoxidative damage (9). Recent studies have identified the existence ofcirculating oxidized LDL (10, 11). These particles have only minimaloxidative damage and are not recognized by scavenger receptors. Bindingof antibodies to these circulating oxidized LDL particles may help toremove them from the circulation before they accumulate in the vasculartissue (12).

Several studies have supported a role for antibodies in protectionagainst atherosclerosis. B cell reconstitution inhibits development ofatherosclerosis in splenectomized apo E null mice (13) as well asneointima formation after carotid injury in RAG-1 mice (unpublishedobservations from our laboratory). Moreover, it has been shown thatrepeated injections of immunoglobulins reduce atherosclerosis in apo Enull mice (6).

As discussed above antibodies against MDA-modified peptide sequences inapo B-100 may be generated by active immunization using syntheticpeptides. This procedure requires 2-3 weeks before a full effect onantibody production is obtained.

In some situations a more rapid effect may be needed. One example may beunstable atherosclerotic plaques in which oxidized LDL is likely tocontribute to inflammation, cell toxicity and risk for plaque rupture.Under these circumstances a passive immunization by injection ofpurified, or recombinantly produced antibodies against native andMDA-modified sequences may have a faster effect.

Another situation in which a passive immunization by injection ofpurified, or recombinantly produced antibodies may be effective iscoronary heart disease in older individuals. Our studies have shown thata decrease in antibodies against apo B peptide sequences occurs withincreasing age in man and is associated with an increase in the plasmalevel of oxidized LDL (Nordin Fredrikson, Hedblad et al). This maysuggest a senescence of the immune cells responsible for producingantibodies against antigens in oxidized LDL and result in a defectiveclearance of oxidatively damaged LDL particles from the circulation.Accordingly, these subjects would benefit more from a passiveimmunization by injection of purified, or recombinantly producedantibodies than from an active immunization with apo B-100 peptidesequences.

Synthetic native peptides (Euro-Diagnostica AB, Malmö, Sweden) used inthe following were peptide 1, 2 and 301 from the initially screenedpolypeptide library. Peptide 1 (amino acid sequence:EEEMLENVSLVCPKDATRFK, n=10; (SEQ ID NO: 15)) and peptide 301 (amino acidsequence: HTFLIYITELLKKLQSTTVM, n=10; (SEQ ID NO: 20)) were found tohave higher IgG or IgM antibody response to MDA modified form thannative peptide, respectively and both titers are higher in healthysubject. These peptides were chosen based on the assumption thatantibody response to these peptides might be protective againstatherosclerosis.

Peptide 2 (amino acid sequence: ATRFKHLRKYTYNYQAQSSS, n=10; (SEQ ID NO:38)) elicited no antibody response in the initial antibody screening,hence it was chosen as control peptide. Mice receiving Alum served ascontrol (n=9).

Apo E (−/−) mice received subcutaneous primary immunization at 6-7 weeksof age, followed by an intra-peritoneal booster 3 weeks later. Mice werefed high cholesterol diet from the onset of immunization and continueduntil sacrifice at the age of 25 weeks. At the time of sacrifice, therewas no significant difference in body weight among 4 groups of mice. Northere was statistically significant difference in serum cholesterol asmeasured using a commercially available kit (Sigma). Their mean serumcholesterol levels were all above 715 mg/dl.

The area of the descending aorta covered by atherosclerotic plaque wasmeasured in an en face preparation after oil red O staining. Incomparison to the control group, mice immunized with peptide No. 2 andNo. 301 had substantially reduced atherosclerosis (FIG. 2). Immunizationwith Peptide No 1 did not produce a significant reduction inatherosclerosis in comparison to control. In contrast to the descendingaorta, extent of atherosclerosis in the aortic root and aortic arch didnot differ among the 4 experimental groups (FIG. 3).

There were no difference among 4 groups in terms of aortic sinus plaquesize or its lipid content (Table A). Mean plaque sizes in the aroticaortic arches from 4 groups of mice were not different. However, en faceevaluation of plaque sizes from descending thoracic and abdominal aortaby oil red O staining revealed that control group and peptide No. 1group had similar amount of atherosclerotic plaque in the aorta, whereaspeptide No. 2 and No. 9 groups had a significantly reducedatherosclerotic burden in the aorta (Table A). The observation thatpeptide immunization did not affect aortic sinus or aortic arch plaquesize but reduced descending aortic plaque is intriguing and suggeststhat peptide immunization might reduce new plaque formation but does notaffect the progression of plaques.

It was further tested whether peptide immunization modulates thephenotype of atherosclerotic plaques. Frozen sections form aortic sinusplaques were immunohistochemically stained with monocyte/macrophageantibody (MOMA-2, Serotec). In concordance with the findings from enface observation, peptide No. 2 significantly reduced macrophageinfiltration in the plaques (FIG. 1). Trichrome staining revealed a meancollage content of 40.0±7.7% in the aortic sinus plaques from peptide 2group; whereas mean collagen content in alum control group, peptide 1group and peptide 9 group were 32.3±5.3%, 35.6±8.5% and 29.4±9.6%,respectively.

Antibody response against immunized peptide in each group wasdetermined. Antibody titer after immunization increased 6.1±3.1 fold inpeptide 1 group, 2.4±1.0 fold in peptide 2 group and 1.8±0.6 fold inpeptide 9 group; whereas alum group had a 3.9±2.7 fold increase ofantibody titer against peptide 1, 2.0±0.5 fold increase against peptide2 and 2.0±0.9 fold increase against peptide 9. It is surprising theparallel increase of antibody titer against immunized peptides both inimmunized and alum treated group. This may mean the followingpossibilities: (1) mechanism(s) other than humoral immune response (suchas cellular immune response) may be involved in modulatingatherosclerosis; or (2) this increase of antibody was a by-standerresponse to hypercholesterolemia with time.

Although there is no clear speculative mechanism to explain why peptideimmunization reduced atherosclerosis and/or modulate plaque phenotype,the novelty of this invention is the concept of using peptides of LDL asimmunogen and its feasibility as an immunomodulation strategy. Thispeptide-based immunization strategy modulates atherosclerotic plaques.Immunization using homologous oxLDL or native LDL as antigen had beenshown to reduce plaque size¹⁻³, however, the availability, production,infectious contamination and safety of homologous human LDL make thisapproach unappealing for clinical application. Here it is demonstratedthat peptide-based immunotherapy is feasible although our final resultsdiffer from our initial hypothesis that immunization using peptides withhigher IgM or IgG antibody response in normal subjects may protectexperimental animals from developing advanced atherosclerotic plaques.

It is surprising to find that immunization using peptide No. 2 protectedanimal from developing new atherosclerotic lesions in descending aortaand reduced macrophage infiltration and a higher collagen content inplaques since this peptide did not render any antibody response frominitial human screen. It may be because (a) peptide No. 2 may be a partof human apo-B-100 protein structure that was not exposed to humanimmune system. Hence, no antibody was generated and detected fromhealthy human serum pools; (b) the amino acid sequence of peptide No. 2is foreign to mice therefore mice developed immune response against thispeptide, which modulates new atherosclerotic lesion formation and itsphenotype.

The effect of homologous LDL immunization on plaque size varied whenplaque sizes were evaluated at different portions of aortic tree. Forexample, Ameli et al showed in hypercholesterolemic rabbit native LDLimmunization resulted in a reduction of plaque formation in aorta¹,whereas Freigang et al. showed reduction of plaque size in aortic sinusbut not in aorta². Taken their findings and the present ones together,it was speculated that peptide immunization modulates not only plaquesizes but also plaque composition. The plaque-reducing effect was onlyobserved in descending aorta. Apo E (−/−) mice are known to developatherosclerotic lesions at various stages of evolution in a singleanimal, especially when fed high cholesterol diet. The initialappearance of atherosclerotic lesion in young animal was in the aorticsinus^(6, 7) and after 15 weeks on high fat-high cholesterol dietlesions at aortic sinus were advanced plaques; whereas earlier stage ofatherosclerosis was present in descending aorta.⁶ Since the temporalcourse of plaque maturation and development in the descending aorta islate compared to that of aortic sinus, the finding that immunizationreduced lesion sizes in the descending aorta but not in aortic sinussuggested immunization affects early stage of atherosclerosis formation.It is possible that as animal aged and in the presence ofsupra-physiological level of serum cholesterol the plaque reducingeffect of immunization is overcome by the toxic effect ofhypercholesterolemia. It is also possible that aortic sinus plaquesmature faster and sacrifice at 25 weeks is too late to detect anydifference in plaque size. Though lesion size was not modulated in theaortic sinus plaque, peptide immunization did modulate plaquecompositions. The present experimental design prevented from studyingthe composition of the plaques in their earlier stage of development indescending aorta.

The experimental findings highlight the feasibility of using peptidesequences of LDL associated apo B-100 as immunogens for a novel approachto preventing atherosclerosis and or favorably modulating plaquephenotype despite severe hyperlipidemia. This peptide-based immunizationstrategy is potentially advantageous over the use of homologous oxLDL ornative LDL as antigen because such a strategy could eliminate the needfor isolation and preparation of homologous LDL and its attendant risksfor contamination. The plaque-reducing effect of immunization withPeptide No 2 and 301 was only observed in descending aorta. Thesefindings are consistent with previous reports where other therapeuticinterventions have also been shown to have a greater effect ondescending aorta compared to the aortic arch¹⁴⁻¹⁷ presumably becauselesions develop more rapidly in the aortic root and the arch than thedescending aorta thus creating a smaller window of opportunity forintervention^(14, 15, 16, 18, 19). Since the temporal course of plaquematuration and development in the descending aorta is late compared tothat of aortic sinus and the aortic arch, the finding that immunizationreduced lesion sizes in the descending aorta but not in aortic sinus andarch suggest that immunization preferentially prevents early stage ofatherosclerosis formation. It is possible that as animal aged and in thepresence of supra-physiological level of serum cholesterol the plaquereducing effect of immunization is overcome by the toxic effect ofsevere hypercholesterolemia. Though the lesion size was not modulated inthe aortic sinus or arch, immunization with Peptide No 2 did modulateplaque composition in a favorable direction creating a more stableplaque phenotype with reduced macrophage infiltration and increasedcollagen content. In summary, it is demonstrated a novel peptide-basedimmunomodulatory approach for inhibition of atherosclerosis in themurine model.

In summary, it is demonstrated a novel peptide-based immunomodulatoryapproach in modulate atherosclerotic plaques. Although the change inatherosclerosis formation in our model was only modest, yet thispeptide-based immunization may provide an alternative tool in studying,preventing or treating atherosclerosis.

Methods

Peptide preparation. Peptides were prepared using Imject® SuperCarrier®EDC kit (Pierce, Rockford, Ill.) according to manufacturer's instructionwith minor modification. One mg peptide in 500 μl conjugation buffer wasmixed with 2 mg carrier in 200 μl deionized water. This mixture was thenincubated with 1 mg conjugation reagent (EDC,1-ethyl-3-[3-dimethylaminopropyl]carbodiimide HCl) in room temperaturefor 2 hours. This was then dialyzed against 0.083 M sodium phosphate,0.9 M sodium chloride pH 7.2 solution overnight at 4° C. The dialyzedconjugate was diluted with Imject dry blend purification buffer to afinal volume of 1.5 ml. Alum was used as immunoadjuvant and mixed withpeptide conjugate with 1:1 dilution in volume. The amount of peptide ineach immunization was 33 μg/100 μl per injection.

Animal protocol. Apo E (−/−) mice from the Jackson Laboratories (BarHarbor, Me.) received subcutaneous primary immunization at 6-7 weeks ofage, followed by an intra-peritoneal booster 3 weeks later. Mice werefed high cholesterol diet from the onset of immunization and continueduntil sacrifice at the age of 25 weeks. Blood samples were collected 2weeks after booster and at the time of sacrifice. Mice receiving Alumserved as control. Experimental protocol was approved by theInstitutional Animal Care and Use Committee of Cedars-Sinai MedicalCenter. All mice were housed in an animal facility accredited by theAmerican Association of Accreditation of Laboratory Animal Care and kepton a 12-hour day/night cycle and had unrestricted access to water andfood. At the time of sacrifice, mice were anesthetized by inhalationEnflurane. Plasma was obtained by retro-orbital bleeding prior tosacrifice.

Tissue harvesting and sectioning. To evaluate the effect of peptideimmunization on atherosclerosis formation, the plaque size at aorticsinus was assessed, aortic arch and descending thoracic and abdominalaorta. After the heart and aortic tree were perfused with normal salineat physiological pressure, the heart and proximal aorta were excised andembedded in OCT compound (Tissue-Tek) and frozen sectioned. Serial6-μm-thick sections were collected from the appearance of at least 2aortic valves to the disappearance of the aortic valve leaflets foraortic sinus plaque evaluation. Typically 3 consecutive sections were onone slide and a total of 25-30 slides were collected from one mouse andevery fifth slide was grouped for staining. Ascending aorta and aorticarches upto left subclavian artery were also sectioned and processedsimilarly. Descending thoracic and abdominal aorta were processedseparately for en face evaluation of plaque formation after oil red Ostaining. En face preparation of descending thoracic and abdominal aorta

Chicken egg albumin (Sigma) in a concentration of 0.8 g/ml water wasmixed 1:1 with glycerol. Sodium azide was added to make a finalconcentration of sodium azide 0.2%. After descending thoracic andabdominal aorta was cleaned off surrounding tissue and fat, the segmentof aorta from left subclavian artery to the level of renal artery wasthen carefully removed for overnight fixation in Histochoice (Amresco).Aorta was then carefully opened longitudinally and placed with luminalside up on a slide freshly coated with egg albumin solution. Afteralbumin solution became dry, the aorta was stained with Oil red O toassess the extent of atherosclerosis with computer-assistedhistomorphometry.

Immunohistochemistry and Histomorphometry. The sections from aorticsinus were immunohistochemically stained with MOMA-2 antibody (Serotec)using standard protocol. Trichrome stain to assess collagen content andoil red O stain for plaque size and lipid content were done usingstandard staining protocol. Computer-assisted morphometric analysis wasperformed to assess histomorphometry as described previously.⁸

Antibody titer measurement. To measure the antibody response afterpeptide immunization, an ELISA was developed. Antibody titer againstimmunized peptide was measured using blood collected at 2 weeks afterbooster and at sacrifice. Antibody response against 3 peptides was alsodetermined in Alum group at the same time-points. In brief, nativesynthetic peptides diluted in PBS pH 7.4 (20 μg/ml) were absorbed tomicrotiter plate wells (Nunc MaxiSorp, Nunc, Roskilde, Denmark) in anovernight incubation at 4° C. After washing with PBS containing 0.05%Tween-20 (PBS-T) the coated plates were blocked with SuperBlock in TBS(Pierce) for 5 min at room temperature followed by an incubation ofmouse serum diluted 1/50 in TBS-0.05% Tween-20 (TBS-T) for 2 h at roomtemperature and then overnight at 4° C. After rinsing, deposition ofantibodies directed to the peptides was detected by using biotinylatedrabbit anti-mouse Ig antibodies (Dako A/S, Glostrup, Denmark)appropriately diluted in TBS-T. After another incubation for 2 h at roomtemperature the plates were washed and the bound biotinylated antibodieswere detected by alkaline phosphatase conjugated streptavidin (Sigma),incubated for 2 h at room temperature. Using phosphatase substrate kit(Pierce) developed the colour reaction and the absorbance at 405 nm wasmeasured after 1 h of incubation at room temperature. Mean values werecalculated after the background was subtracted.

Other assay models is of course applicable as well, such any immunoassaydetecting an antibody, such as radioactive immunoassay, Westernblotting, and Southern blotting, as well as detection of antibodiesbound to peptides, enzyme electrodes and other methods for analysis.

Statistics

Data are presented as mean±Std. Statistical method used is listed ineither text, table or figure legend. P<0.05 was considered asstatistically significant.

TABLE A Aortic sinus plaque size and its lipid content, aortic archplaque size and percent of plaque in descending aorta. Oil red O (+)area Total plaque size in (% of aortic sinus Aortic arch plaque % ofplaque in aortic sinus (mm²) plaque) size (mm²) aorta (flat prep.) Alum0.49 ± 0.13 21.7 ± 4.4 0.057 ± 0.040 20 ± 4.7 Peptide 1 0.48 ± 0.14 32.0± 8.1 0.054 ± 0.027 17 ± 4.3 Peptide 301 0.46 ± 0.16 23.8 ± 4.1 0.050 ±0.024  8.9 ± 2.2* *Significant different from Alum group. ANOVA followedby Tukey-Kramer test was used for statistical analysis.

Further data on the effect of immunization with apolipoprotein B-100peptide sequences on atherosclerosis in apo E knockout mice is givenbelow in Table B

TABLE B Effect of immunization with apolipoprotein B-100 peptidesequences on atherosclerosis in apo E knockout mice Effect onatherosclerosis in the aorta Immunizations using mixtures of severalpeptide sequences 1. Peptide sequences 143 and 210 −64.6% 2. Peptidesequences 11, 25 and 74 −59.6% 3. Peptide sequences 129, 148 and 167−56.8% 4. Peptide sequences 99, 100, 102, 103 and 105 −40.1% 5. Peptidesequences 30, 31, 32, 33 and 34  +6.6% 6. Peptide sequences 10, 45, 154,199 and 240 +17.8% Immunizations using a single peptide sequence 1.Peptide sequence 2 −67.7% 2. Peptide sequence 210 −57.9% 3. Peptidesequence 301 −55.2% 4. Peptide sequence 45 −47.4% 5. Peptide sequence 74−31.0% 6. Peptide sequence 1 −15.4% 7. Peptide sequence 240     0%

Administration of the peptides is normally carried by injection, such assubcutaneous injection, intravenous injection, intramuscular injectionor intraperitoneal injection. A first immunizing dosage can be 1 to 100mg per patient depending on body weight, age, and other physical andmedical conditions. In particular situations a local administration of asolution containing one or more of the peptides via catheter to thecoronary vessels is possible as well. Oral preparations may becontemplated as well, although particular precautions must be taken toadmit absorption into the blood stream. An injection dosage may contain0.5 to 99.5% by weight of one or more of the fragments or peptides ofthe present invention.

The peptides are normally administered as linked to cationized bovineserum albumine, and using aluminium hydroxide as an adjuvant. Otheradjuvants known in the art can be used as well.

Solutions for administration of the peptides shall not contain any EDTAor antioxidants.

The peptides can also be used as therapeutic agents in patients alreadysuffering from an atheroschlerosis. Thus any suitable administrationroute can be used for adding one or more of the fragments or peptides ofthe invention.

Initial studies focused on determining which type of oxidativemodifications of peptides led to recognition by antibodies in humanplasma. These studies were done using peptides 1-5 and 297-302. Duringoxidation of LDL polyunsaturated fatty acids in phospholipids andcholesteryl esters undergo peroxidation leading to formation of highlyreactive breakdown products, such as malondealdehyde (MDA). MDA may thenform covalent adducts with lysine and histidine residues in apo B-100making them highly immunogenic. Oxidation of LDL also results infragmentation of apo B-100 that may lead to exposure of peptidesequences not normally accessible for the immune system. In theseexperiments peptides were used in their native state, after MDAmodification or after incorporation into phospholipid liposomes followedby copper oxidation or MDA-modification. IgM antibodies were identifiedagainst native, MDA- and liposome oxidized peptides, with antibodytiters MDA-peptide>MDA-modified liposome peptides>liposome oxidizedpeptide>native peptide. Specificity testing demonstrated that binding ofantibodies to MDA-modified peptides was competed by both MDA-LDL andcopper oxidized LDL.

We then performed a screening of the complete peptide library usingpooled plasma derived from healthy control subjects and native andMDA-modified peptides as antigens. Antibodies to a large number of sitesin apo B-100 were identified. Using twice the absorbance of thebackground control as positive titer cut off, antibodies were detectedagainst 102 of the 302 peptides constituting the complete apo B-100sequence. IgM binding was substantially more abundant than that of IgG.Generally, binding was higher to MDA modified peptide sequences than tothe corresponding native sequence, but these was a striking correlationbetween the two. Binding to both native and MDA modified sequences wascompeted by addition of MDA-modified LDL and copper oxidized LDL, butnot by native LDL. These observations suggest that immune responsesagainst MDA-modified peptide sequences in apo B-100 results in a crossreactivity against native sequences. The inability of native LDL tocompete antibody binding to native apo B-100 peptide sequences isintriguing, but may indicate that these sequences only become exposedafter the proteolytic degradation of apo B-100 that occurs as a resultof LDL oxidation. Both hydrophilic and hydrophobic parts of the moleculewere recognized by antibodies. A second screening of the apo B-100peptide library was performed using pooled plasma from subjects withclinical signs of coronary heart disease (CHD, acute myocardialinfarction (AMI) and unstable angina; n=10). Antibodies in pooled CHDplasma bound to the same sequences and with the same overalldistribution as for antibodies in healthy control plasma. However,antibody titers to several peptides (#1, 30-34, 100, 107, 148, 149, 162,169, 236, 252 and 301) were at least twice as high as in control plasmacompared to plasma from CHD subjects, whereas titers against a fewpeptides (#10, 45, 111, 154, 199, 222 and 240) were higher in plasmafrom CHD patients compared to controls. We then performed a prospectiveclinical study to investigate if antibody levels against MDA-modifiedpeptide sequences in apo B-100 predict risk for development of CHD.Using a nested case control design we selected 78 subjects with coronaryevents (AMI or death due to CHD) and 149 controls from the Malmö DietCancer Study. Neither cases nor control individuals had a history ofprevious MI or stroke. The median time from inclusion to the acutecoronary event was 2.8 years (range 0.1-5.9 years) among cases. Antibodylevels were determined in baseline plasma samples supplemented withantioxidants. Using the carotid intima-media thickness (IMT) as assessedby ultrasonography at baseline we also analyzed associations betweenantibody levels and degree of existing vascular disease. We studied 8MDA-modified peptide sequences that in the initial screening studieswere associated with high plasma antibody levels (#74, 102 and 210)and/or marked differences between control and CHD plasma pools (#32, 45,129, 162 and 240). Controls were found to have higher IgM levels againstMDA peptide 74 (0.258, range 0-1.123 absorbance units versus 0.178,range 0-0.732 absorbance units, p<0.05), otherwise there were nodifferences in antibody levels between cases and controls. Associationsbetween IMT and IgM against MDA-peptides #102, 129, and 162 (r=0.233,0.232, and 0.234, respectively, p<0.05) were observed in cases andbetween IMT and MDA-peptide 45 (r=0.18, p<0.05) in controls. Weakcorrelations were observed between antibodies to MDA peptide 129 andtotal and LDL cholesterol (r=0.19 and r=0.19, p<0.01, respectively),otherwise peptide antibody levels showed no associations with totalplasma cholesterol, LDL cholesterol, HDL cholesterol or plasmatriglycerides. There were strong co-variations between antibody levelsto the different peptides (r values ranging from 0.6 to 0.9). The onlyexception was antibodies against MDA-peptide 74 that were weakly or notat all related to antibodies against the other peptides.

Antibodies against all sequences except MDA-peptide 74 was inverselyassociated with age among cases (r values ranging from −0.38 to −0.58,p<0.010.001), but not in controls. Plasma levels of oxidized LDL, incontrast, increased with age. Again this association was stronger incases than in controls. To investigate if the associations betweenimmune responses against MDA-modified peptide sequences andcardiovascular disease were different in different age groups a subgroupanalysis was performed on cases and controls under and above the medianage (61 years). In the younger age group cases had increased antibodylevels against peptides 32 and 45 and decreased antibody levels againstpeptide 74 as compared to controls, whereas no differences were seen inthe older age group. Antibodies against all MDA peptide sequences,except peptide 74, were significantly associated with IMT in the youngerage group, but not in the older (Table).

These studies identify a number of MDA-modified sequences in apo B-100that are recognized by human antibodies. MDA-modification of apo B-100occurs as a result of LDL oxidation indicating that these antibodiesbelong to the family of previously described oxidized LDLautoantibodies. This notion is also supported by the observation thatantibody binding to MDA-modified apo B-100 peptides is competed byaddition of oxidized LDL. Together with the oxidized phospholipidsidentified by Hörkkö et al, these MDA-modified peptide sequences arelikely to constitute the large majority of antigenic structures inoxidized LDL. In similarity with the oxidized LDL antiphospholipidantibodies, antibodies against MDA-modified apo B-100 sequences were ofIgM type. This may suggest that also the latter antibodies belong to thefamily of T 15 natural antibodies. T 15 antibodies have been attributedan important role in the early, T cell independent defense againstbacterial infections as well as in the removal of apoptotic cells. Itremains to be determined if the MDA-peptide antibodies described herehave similar functions. Antibodies were also identified against a largenumber of native apo B-100 sequences. However, the striking co-variationbetween antibodies to native and MDA-modified sequences suggests thatalso these antibodies are formed in response to LDL oxidation. It isalso possible that antibodies against an MDA-modified peptide sequencecross reacts with the corresponding native sequence. If antibodiesagainst native apo B-100 sequences bind also to native LDL particlesthis is likely to have a major influence on LDL metabolism. However, thefinding that native LDL does not compete antibody binding to native apoB-100 sequences, as well as the lack of correlation between antibodiesagainst native apo B-100 sequences and LDL cholesterol levels againstthe existence of such a phenomena.

Antibodies against MDA-modified peptide sequences decreasedprogressively with age in the cases, but not in the controls. With theexception of MDA-peptide 74, IgM antibodies against MDA-peptides weresignificantly associated with carotid IMT in the younger age group(below 62 years), but not in the older age group. These findings suggestthat significant changes in the interactions between the immune systemand the atherosclerotic vascular wall takes place between ages 50 and 70years. One possibility is that in younger individuals theatherosclerotic disease process is at a more active stage with a moreprominent involvement of immune cells. Another possibility is that thedecreased levels of antibodies against MDA-modified peptide sequences inolder subjects reflect a senescence of the immune cells involved inatherosclerosis. An impaired function of immune cells due toimmunosenescence have been proposed to contribute to an increasedsusceptibility to infection and cancer in the older population.Interestingly, immunosenescence is inhibited by antioxidants indicatinginvolvement of oxidative stress. Immune cells that interact withepitopes in oxidized LDL are likely to be particularly exposed tooxidative stress. Since oxidized LDL is present in arteries already at avery early age these immune response are being continuously challengedfor several decades, which may further contribute to a development ofimmunosenescence.

Increased antibodies against two sites in apo B-100 were found topredict risk for myocardial infarction and coronary death in subjectsbelow 62 years of age. Antibodies against these sites showed a highlevel of co-variation suggesting that they were produced in response tothe same underlying pathophysiological processes. The fact that themedian time from blood sampling to coronary event was only 2.8 yearsmakes these antibodies particularly interesting as makers for increasedCHD risk. Antibody levels against MDA-modified apo B-100 peptidesequences showed no associations with other CHD risk factors such ashyperlipidemia, hypertension and diabetes suggesting that they areindependent markers of CHD risk. The CHD cases in the present study werenot extremely high-risk individuals and in this respect representativeof the common CHD patient. The finding that IgM against MDA-modified apoB-100 sequences predicts short-term risk for development of acutecoronary events in individuals that would not have been identified ashigh risk by screening of established risk factors suggest that it maybecome a useful instrument in identifying individuals in need ofaggressive preventive treatment. However, considerably largerprospective studies with multivariate analysis are required before theclinical value of determining antibodies against apo B-100 MDA-modifiedpeptide sequences can be fully established. Another limitation of thepresent clinical study is that we have only analysed antibodies againsta small number of the antigenic sites in apo B-100 and that antibodytiters against other sites may be even better markers of cardiovascularrisk.

In subjects below age 60 antibodies against a large number ofMDA-modified sites in apo B-100 were correlated with the extent ofexisting vascular disease as assessed by carotid IMT. IgM antibodieswere more closely associated with carotid IMT than IgG antibodies.Although carotid IMT has obvious limitations as a measure of generalatherosclerotic burden these observations still suggest thatdetermination of IgM against MDA-modified sequences in apo B-100 may beone method to assess the severity of existing atherosclerosis. Theseobservations are also in line with several previous studies that havereported associations between coronary and carotid artery disease andIgM antibodies against oxidized LDL.

Antibodies against peptide 74 differed against other apo B-100 peptideantibodies in many respect. They were higher in controls than in cases,they did not decrease with age and were not associated with the extentof carotid disease. Accordingly, antibodies against this peptidesequence represent interesting candidates for an athero-protectiveimmune response.

An important question is why these associations occur. They clearlydemonstrate that immune responses against MDA-modified apo B-100 sitessomehow are involved in the atherosclerosic disease process. Since highantibody levels are associated with more severe atherosclerosis andincreased risk for development of acute coronary events one obviouspossibility is that these immune responses promote atherogenesis.Studies demonstrating that immune responses against heat shock proteins,such as HSP 65, are atherogenic provide some support for this notion.However, experimental animal studies have shown an athero-protectiveeffect of oxidized LDL immunization. B cell reconstitution of spleenectomized apo E null mice results in a decrease in atherosclerosis.Reduced atherosclerosis has also been observed in apo E null mice givenrepeated injections of immunoglobulin. The present observations do notnecessarily argue against an athero-protective role of immune responsesagainst oxidized LDL. These immune responses are activated bypro-atherogenic processes such as LDL oxidation. Accordingly, they arealso likely to be in proportion to the severity of the disease processand could serve as makers of disease severity and CHD risk withoutcontributing to disease progression. The finding that immunization ofapo E null mice with apo B-100 peptide sequences inhibits development ofatherosclerosis reported in two accompanying papers demonstrates thatthis is likely to be the case. Indeed, the most important outcome of thepresent study may well be the identification of structures that could beused as components of a vaccine against atherosclerosis. The observationthat the decrease in antibodies against MDA-modified peptide sequencesin apo B-100 that occurs with age is accompanied by an increase inplasma levels of oxidized LDL suggest that an increased clearance ofminimally oxidized LDL from the circulation may be one mechanism bywhich these antibodies could protect against atherosclerosis.

Methods Study Population

The study subjects, borr between 1926-45, belong to the Malmö “Diet andCancer (MDC)” study cohort. A random 50% of those who entered the MDCstudy between November 1991 and February 1994 were invited to take partin a study on the epidemiology of carotid artery disease. Routines forascertainment of information on morbidity and mortality following thehealth examination, as well as definition of traditional risk factors,have been reported.

Eighty-five cases of acute coronary heart events, i.e. fatal ornon-fatal MI or deaths due to coronary heart disease (CHD) wereidentified. Participants who had a history of myocardial infarction orstroke (n=6) were not eligible for the present study. For each case twocontrols without a history of myocardial infarction or stroke wasindividually matched for age, sex, smoking habits, presence ofhypertension and month of participation in the screening examination andduration of follow-up. Due to logistic reason (blood samples were notavailable in sufficient quantity for assessment of peptides) only onecontrol was available for seven cases and no controls for one case. Thiscase was excluded from analysis. Thus the study population consists of227 subjects, 78 cases and 149 controls, aged 49-67 (median 61) years atbaseline.

Laboratory Analyses

After overnight fasting blood samples were drawn for the determinationof serum values of total cholesterol, triglycerides, HDL cholesterol,LDL cholesterol and whole blood glucose. LDL cholesterol in mmol/L wascalculated according to the Friedewald formula. Oxidized LDL wasmeasured by ELISA (Mercordia).

B-Mode ultrasound Vasculography

An Acuson 128 Computed Tomography System (Acuson, Mountain View, Calif.)with a MHz transducer was used for the assessment of carotid plaques inthe right carotid artery as described previously.

Development of ELISAs Against Apo B-100 Peptide Sequences

The 302 peptides corresponding to the entire human apolipoprotein Bamino acid sequence were synthesized (Euro-Diagnostica AB, Malmö, Swedenand K J Ross Petersen A S, Horsholm, Denmark) and used in ELISA. Afraction of each synthetic peptide was modified by 0.5 M MDA(Sigma-Aldrich Sweden AB, Stockholm, Sweden) for 3 h at 37° C. and inpresence of liposomes by 0.5 M MDA for 3 h at 37° C. or by 5 mM CUCl₂(Sigma) for 18 h at 37° C. The MDA modified peptides were dialysedagainst PBS containing 1 mM EDTA with several changes for 18 h at 4° C.The modification of the peptides was tested in denatured polyacrylamidegels (BioRad Laboratories, Hercules, Calif.), suitable for separation ofpeptides.

A mixture of egg phosphatidylcholine (EPC) (Sigma) andphosphatidylserine (PS) (Sigma) in a chloroform solution at a molarratio of 9:1 and a concentration of 3 mM phospholipid (PL) wasevaporated in a glass container under gentle argon stream. The containerwas then placed under vacuum for 3 hours. A solution containing 0.10 mMpeptide (5 ml) in sterile filtered 10 mM HEPES buffer pH 7.4, 145 mMNaCl and 0.003% sodium azide was added to the EPC/PS dried film andincubated for 15 min at 50° C. The mixture was gently vortex for about 5min at room temperature and then placed in ice-cold water bath andsonicated with 7.5 amplitude microns for 3×3 min (Sonyprep 150 MSESanyo, Tamro-Medlab, Sweden) with 1 min interruptions. The PL-peptidemixture, native or modified by 0.5 M MDA for 311 at 37° C. or 5 mM CUCl₂for 18 h at 37° C., was stored under argon in glass vials at 4° C.wrapped in aluminum foil and used within 1 week. The MDA-modifiedmixture was dialyzed against PBS containing 1 mM EDTA with severalchanges for 18 h at 4° C. before storage. The modification of themixture was tested in denatured polyacrylamide gels (BioRad LaboratoriesAB; Sundbyberg, SE), suitable for separation of peptides.

Native or modified synthetic peptides diluted in PBS pH 7.4 (20 leg/ml),in presence or absence of liposomes, were absorbed to microtiter platewells (Nunc MaxiSorp, Nunc, Roskilde, Denmark) in an overnightincubation at 4° C. As a reference, one of the peptides (P6) was ran oneach plate. After washing with PBS containing 0.05% Tween-20 (PBS-T) thecoated plates were blocked with SuperBlock in TBS (Pierce, Rockford,Ill.) for 5 min at room temperature followed by an incubation of pooledhuman plasma, diluted 1/100 in TBS-0.05% Tween-20 (TBS-T) for 2 h atroom temperature and then overnight at 4° C. After rinsing, depositionof auto-antibodies directed to the peptides were detected by usingbiotinylated rabbit anti-human IgG- or IgM-antibodies (Dako A/S,Glostrup, Denmark) appropriately diluted in TBS-T. After anotherincubation for 2 h at room temperature the plates were washed and thebound biotinylated antibodies were detected by alkaline phosphataseconjugated streptavidin (Sigma), incubated for 2 h at room temperature.The color reaction was developed by using phosphatase substrate kit(Pierce) and the absorbance at 405 nm was measured after 1 h ofincubation at room temperature. The absorbance values of the differentpeptides were divided with the absorbance value of P6 and compared.

Statistics

SPSS was used for the statistical analyses. The results are presented asmedian and range and as proportions when appropriate. Boxplot andscatterplots were used till illustrate the relationship between age andselected peptides among cases and corresponding controls. Correspondinggraphs were also used to illustrate the relationship between age andselected peptides, cases and controls, respectively, below and above themedian age (61 year) at baseline and separately for cases and controlsbelow the median age. In cases and controls, separately, partialcorrelation coefficients, adjusted for age and sex, were computedbetween selected peptides and blood lipid levels and common carotid IMT.Age- and sex adjusted partial correlation coefficients were alsocomputed between common carotid IMT and selected peptides in cases andcontrols below and over the median age. An independent sample t-test wasused to assess normally distributed continuous variables and aChi-square test for proportions between cases and controls.Non-parametric test (Mann-Whitney) was used to assess non-normallydistributed continuous variables between cases and controls. Allp-values are two-tailed.

TABLE Age- and sex adjusted correlation coefficient for differentbaseline MDA peptides and common carotid artery intima-media thicknessamong younger (49-61 years) and older (62-67 years) cases withmyocardial infarction and their corresponding controls matched for age,sex, smoking and hypertension. CASES plus CONTROLS CASES plus CONTROLSPEPTIDE Aged 49-61 year, n = 116 Aged 62-67 year, n = 111 IGM MDA 320.235t −0.101 MDA 45 0.366$ −0.030 MDA 74 0.178 0.063 MDA 102 0.255$−0.039 MDA 129 0.330$ −0.009 MDA 162 0.2451 0.001 MDA 210 0.254 0.013MDA 240 0.284$ 0.006 IGG MDA 215 0.119 −0.059 p < 0.05; $/×0.01

1-13. (canceled)
 14. A pharmaceutical preparation comprising atherapeutically effective amount of the apo B-100 peptide fragments SEQ.ID. NO. 9, SEQ. ID. NO. 14, SEQ. ID. NO. 32, and SEQ. ID. NO. 38,optionally in combination with one or more pharmaceutically innocuousfillers, or adjuvants, or both pharmaceutically innocuous fillers andadjuvants.
 15. The pharmaceutical preparation according to claim 14,wherein the fragments are present as linked to cationized bovine serumalbumin, said preparation comprising aluminum hydroxide as an adjuvant.16. The pharmaceutical preparation according to claim 15, wherein thereparation is injectable.
 17. A vaccine for immunization of mammalsagainst ischemic cardiovascular diseases, said vaccine comprising one ormore fragments of claim 14, optionally in combination with an adjuvant.18. (canceled)
 19. A vaccine according to claim 17, wherein the fragmentis linked to cationized bovine serum albumin, said vaccine comprisingaluminum hydroxide as an adjuvant.
 20. A method of prophylactic ortherapeutic treatment of a mammal suffering from atherosclerosis orfacing the risk of developing ischemic cardiovascular disease, saidmethod comprising administering a therapeutically effective amount ofone or more of the fragments of claim 14 to said mammal.
 21. A method ofprophylactic or therapeutic treatment of a mammal suffering fromatherosclerosis or facing the risk of developing ischemic cardiovasculardiseases, said method comprising administering to the mammal atherapeutically effective amount of one or more purified orrecombinantly produced antibodies against native and MDA-modifiedsequences to produce a passive immunization.
 22. The method according toclaim 20, wherein said atherosclerosis or ischemic cardiovasculardisease is characterized by one or more unstable atherosclerotic plaquesin which oxidized LDL is likely to contribute to inflammation, celltoxicity, and risk for plaque rupture, and coronary heart disease inolder individuals. 23-36. (canceled)